The Effect of Deoxycholate on Cytochrome Oxidase.

Sodium deoxycholate and sodium cholate have been widely used to solubiliie mitochondrial enzymes ever since their use by Keilin and Hartree for clarifying heart muscle preparations (l), by Hopkins, Lutwak-Mann, and Morgan (2) for extracting succinic dehydrogenase, by von Euler and Hellstrom (3) for preparing cytochrome b and cytochrome oxidase (cytochrome c:Os oxidoreductase, EC 1.9.3.1), and by Yakushiji and Okunuki (4) for preparing cytochrome cl. The first highly active soluble form of cytochrome oxidase was obtained with the aid of deoxycholate in 1947 (5, 6), and since that time many investigators have used both deoxycholate (e.g.(7-10)) and cholate (e.g.@-13)) to extract cytochrome b and cytochrome cl as well as cytochrome oxidase. The ability of the bile salts to solubilize the cytochromes is complicated by the fact that these reagents may also modify enzyme activity; under certain conditions both deoxycholate and cholate activate cytochrome oxidase (e.g.(ll, 14-19)), whereas under other conditions they are powerful inhibitors (e.g.(8, 11, 15-24)). Nevertheless, despite their widespread use, few attempts have been made to determine the mechanism of these effects. In 1955 Smith (16) suggested, on the basis of spectrophotometric studies, that cholate probably inhibits cytochrome oxidase by interfering with its reduction by cytochrome c rather than its reoxidation by oxygen. On the other hand, Martin and Stotz (23) reported in 1960 that both reduction and reoxidation of cytochrome oxidase were inhibited by cholate, although the combination of cytochrome oxidase with oxygen was not affected. To our knowledge, similar studies with deoxycholate have not been reported. Our own work on this problem stems from the observation that incubation of a deoxycholate-solubilized preparation of cytochrome oxidase for 30 minutes at 37” markedly inactivated the enzyme. Subsequent studies showed that the inactivation was caused by the deoxycholate present in the cytochrome oxidase preparation; the biie salt apparently converted some of the cytochrome oxidase to a denatured form which was not reducible by its natural substrate, reduced cytochrome c. Furthermore, the spectrum obtained after reduction of the denatured enzyme with dithionite diiered from that of the normal enzyme. Incubation of reduced cytochrome oxidase under the same conditions did not modify the spectrum, nor did it prevent reoxidation of the enzyme by oxygen.